PROJECT SUMMARY/ABSTRACT Interleukin-9 (IL-9) is a pleuripotent cytokine that leads to altered gene expression and cellular function in hematopoietic and structural cell populations. IL-9 is most efficiently produced by innate lymphoid cells, and a specialized subset of T helper cells termed Th9 cells. Importantly, IL-9 has a role, in both humans and mouse models, in asthma, food allergy, ulcerative colitis, and anti-tumor immunity. Using Th9 cells as a model for understanding the regulation of the Il9 gene, we and several other laboratories have identified transcription factors that are important for the expression of the Il9 locus. Yet, there is still very little understood about Il9 regulatory elements and that knowledge will not only provide insight into how the gene is transcribed, but also in providing a framework for determining how human polymorphisms might impact gene expression, immunity, and disease susceptibility. In this small project we will define the function of a putative Il9 regulatory element, termed CNS-25, in both murine and human cells that we identified using bioinformatics approaches. In Aim 1 we will take several approaches to characterizing CNS-25 including p300 ChIP-seq in mouse Th9 cells, performing ChIP assays in human Th subsets, and using CRISPR-Cas9 in primary human T cells to mediate mutation and Cas9-p300-directed transactivation of the locus. In the second Aim we will utilize already- generated mice that lack CNS-25 to define the requirement for this element in Th9 cells, and extend those studies to other IL-9-expressing cells including NKT cells and innate lymphoid cells. We will further test the role of CNS-25 in controlling IL-9-dependent disease in a model of allergic lung inflammation. Together, these Aims will define new mechanisms of IL-9 regulation and provide potential targets for understanding the regulation of this important cytokine in human immune responses.